Orphan receptor

ABSTRACT

This invention relates to a novel estrogen receptor-related nuclear receptor, hereinafter termed “ERβ” having the amino acid sequence of FIGS.  1, 13 A or  14 A or substantially the same amino acid sequence as the amino acid sequence shown in FIGS.  1, 13 A or  13 B or an amino acid sequence functionally similar to that sequence. The invention also relates to DNA sequences encoding the receptor. The receptor may be useful in isolating molecules for the treatment of disorders such as prostate cancer, benign prostatic hyperplasia, osteoporosis or cardiovascular disorders and in the testing of substances for estrogenic and other hormonal effects.

[0001] This invention relates to cellular nuclear receptors and theiruses.

[0002] A large family of nuclear receptors which confer cells withresponsiveness to molecules such as retinoid acid, vitamin D, steroidhormones and thyroid hormones has been identified. Extensive studieshave shown that the members of this superfamily of nuclear receptorsactivate and/or repress gene transcription through direct binding todiscrete cis-acting elements termed “hormone response elements” (HRE).It has been shown that these HRE's comprise repeats of consensuspalindromic hexanucleotide DNA motifs. The specificity of the HRE's isdetermined by the orientation of, and spacing between, halfsites (i.e.half a palindromic sequence)(Umenesono K., et al, 1991 Cell 65,1255-1266).

[0003] Specific DNA binding is mediated by a strongly-conserved DNAbinding domain, containing two zinc fingers, which is conserved amongall thus discovered nuclear receptors. Three amino acids at theC-terminal base of the first zinc finger (known as the “P-box”) areimportant for the recognition of the half site nucleotide sequence.Members of the nuclear receptor superfamily have been classified intodifferent groups on the basis of the amino acid sequence within the Pbox.

[0004] All members of the nuclear receptor superfamily also contain ahypervariable N-terminal domain and a ligand-binding domain containingsome “patches” of conserved sequence. One of these is called the“Ti-domain”.

[0005] Molecules which are thought to be nuclear receptors, as they arestructurally related to characterised receptors, but for which no ligandhas been found, are termed “orphan receptors”. Many such orphanreceptors have been identified (see for example Evans R. M. (1988)Science 240, 889-895 and O'Malley, B. (1990) Mol. Endocrinol. 4 363-369)

[0006] We have now unexpectedly identified, initially in rat a neworphan receptor, which is related to the known estrogen receptor ERα,and which we have designated “ERβ” (specifically “rERβ” in rat). In thisspecification “Erβ” will be used to refer to the receptors hERβ or rERβor related receptors. The nucleotide and amino acid sequences of rERβhave now been determined and are shown in FIG. 1. We have alsoidentified a human Erβ-“hERβ”, the amino acid DNA and sequences of whichare shown in FIGS. 13A and 13B respectively.

[0007] According to one aspect of the invention there is provided anovel estrogen receptor-related nuclear receptor, hereinafter termed“ERβ” having the amino acid sequence of FIG. 1, FIGS. 13A or 16A orsubstantially the same amino acid sequence as the amino acid sequenceshown in FIGS. 1, 13A or 16A or an amino acid sequence functionallysimilar to those sequence. The isolated receptor may be particularlyuseful in the search for molecules for use in treatment of diseases orconditions such as cardiovascular diseases, central nervous systemdiseases or conditions or osteoporosis, prostate cancer or benignprostatic hyperplasia.

[0008] The receptor of the invention may also be used in the testing ofenvironmental chemicals for estrogenic activity. There has beenincreasing concern over the effect of various chemicals released intothe environment on the reproduction of humans and animals. Threats tothe reproductive capabilities of birds, fish, reptiles, and some mammalshave become evident and similar effects in humans have been proposed.Substantial evidence is now emerging which shows that exposure tocertain chemicals during critical periods of foetal life may distort thedevelopment of the reproductive organs and the immune and nervoussystems. On the basis of possible parallels between actual wildlifeeffects, seen for example in birds and seals living in highly pollutedareas, and proposed effects in humans, in combination with documentedhuman reproductive effects caused by prenatal exposure to thepharmaceutical estrogen, diethyl stilbestrol (DES), “estrogenic”chemicals have been proposed to threaten the reproductive capability ofboth animals and humans. Among the chemicals known or suspected to actas estrogen mimics on the human body, or in other ways disturb the humanendocrine system, there are several which have already been identifiedas environmental hazards. Among the chemicals that have been mentionedas potential causes of disruption of reproductive function in animalsand humans are chlorinated organic compounds such dieldrin, endosulfans,chlordanes, endrins, aldrin, DDT and some PCBs, plastics such asBisphenol A, phthalates and nonylphenol, and aromatic hydrocarbons. Someof the proposed effects on humans have been suggested to be due to anincreasing exposure to environmental estrogens—in fact, exposure tochemical compounds to which higher organisms during the foetal periodreact in a way that is similar to when they are exposed to high dosagesof estrogens. The effects are manifested by for example perturbations ofthe sex characteristics and impaired reproductive potential. In humans,elevated risks of breast cancer and other hormone-related disease hasalso been discussed as possible effects. In addition, to the documented“estrogenic” effects, it has recently been demonstrated thatenvironmental pollutants may also act on hormonal pathways other thanthe estrogenic pathway—it has been shown that p,p′—DDE the mainmetabolite of DDT (also in humans) is a fairly anti-androgenic agent(Kelce W. R. et al Nature 1995 375:581-585). Epidemiological studies onthese issues are, however, presently difficult to interpret.Nevertheless, there is a growing opinion against these potentiallyhormone disrupting chemicals, and very palpable public and environmentaldemand for the governmental agencies and industry to act. In view of thesimilarities between the receptor of the present invention, Erβ and theclassical estrogen receptor, Erβ may be used in the testing of chemicalsfor estrogenic effect.

[0009] An amino acid sequence functionally-similar to the sequence shownin FIGS. 1, 13A or 14A may be from a different mammalian species.

[0010] An amino acid sequence which is more than about 89%, identicalwith the sequence shown in FIGS. 1, 13A or 14A is substantially the sameamino acid sequence for the purposes of the present application.Preferably, the amino acid sequences is more than about 95% identicalwith the sequence shown in FIGS. 1, 13A or 14A.

[0011] According to another aspect of the invention there is provided aDNA sequence encoding a nuclear receptor according to the first aspectof the invention. Preferably, the DNA sequence is that given in FIGS. 1,13A or 14A or is a DNA sequence encoding a protein or polypeptide havingthe functionality of ERβ.

[0012] ERβ is unique in that it is extremely homologous to the ratestrogen receptor, in particular in its DNA binding domain. It appearsthat ERβ has a very limited tissue distribution. In female rats, itappears to be present only in the ovaries, and in male rats in theprostate and testes. As these tissues are classic targets for estrogenaction, it can be deduced that ERβ may mediate some of the effects ofestrogen.

[0013] The different ligand specificity of ERα and ERβ may be exploitedto design pharmaceutical agents which are selective for either receptor.In particular, the differences in ligand specificity may be used todevelop drugs that specifically target cardiovascular disease inpostmenopausal women or osteoporosis.

[0014] The nuclear receptor of the invention, ERβ, a method of producingit, and tests on its functionality will now be described, by way ofexample only, with reference to the accompanying drawings, FIGS. 1 to 15in which:

[0015]FIG. 1 shows the amino acid sequence of ERβ and the nucleotidesequence of the gene encoding it;

[0016]FIG. 2A is a phylogenetic tree showing the evolution of ERβ andother receptors;

[0017]FIG. 2B shows the homology between the different domains in ERβand certain other receptors;

[0018]FIG. 2C is an alignment of the amino acid sequence in the ligandbinding domains of rERβ, rERα, mERα and hERα;

[0019]FIG. 2D is an alignment of the amino acid sequence in the DNAbinding domains of rERβ, rERα, mERα and hERα;

[0020]FIG. 3A is a film autoradiograph of prostate gland showing strongexpression of a clone of the receptor of the invention, clone 29;

[0021]FIG. 3B is a darkfield image showing prominent signal for clone 29in epithelium (e) of prostatic alveoli. The stroma(s) exhibit(s) weakersignal;

[0022]FIG. 3C is a bipolarization image of cresyl violet counterstainedsection showing silver grains over epithelium (e), whereas the stroma(s)contain(s) less grains;

[0023] The bar represents 0.7 mm for FIG. 3A, 200 μm for FIG. 3B and 30μm for FIG. 3C;

[0024]FIG. 4A shows a film autoradiograph of ovary showing strongexpression of clone 29 in follicles at different developmental stages(some are indicated by arrows). The interstitial tissue (arrowheads)shows low signal;

[0025]FIG. 4B shows a darkfield image showing high expression of clone29 in granular cells of primary (1), secondary (2), tertiary (3) andmature (4) follicles. Low signal is present in interstitial tissue (it);

[0026]FIG. 4C is a bipolarization image of ovary a showing strong signalin granular cells (gc), whereas the oocyte (oc) and the cainterna (ti)are devoid of clear signal;

[0027] The bar represents 0.9 mm for FIG. 4A, 140 μm for FIG. 4B and 50μm for FIG. 4C;

[0028]FIG. 5A illustrates the results of saturation ligand bindinganalysis of cloned ERβ;

[0029]FIG. 5B illustrates the specificity of ligand binding by clonedERβ;

[0030]FIG. 5C illustrates E2 binding by ERβ;

[0031]FIG. 6 illustrates the activation of transcription by cloned ERβ;

[0032]FIGS. 7 and 7A illustrates stimulation by various ligands bycloned ERβ;

[0033]FIG. 8 illustrates the results of RT-PCR experiments on theexpression of rat estrogen receptors;

[0034]FIG. 9 illustrates the results of RT-PCR experiments on theexpression of human Erβ (hERβ);

[0035]FIG. 10A is a Hill plot comparing binding of ¹²⁵I-E2 by hERα andrERβ;

[0036]FIG. 10B is a Scatchard plot comparing binding of ¹²⁵I-E2 by hERαand rERβ;

[0037]FIG. 11A illustrates the relative binding affinity of hERα andrERβ for various ligands;

[0038]FIG. 11B is a detail of FIG. 12A;

[0039]FIG. 12 is an alignment of various estrogen receptors;

[0040]FIG. 13A shows the amino acid sequence of human ERβ;

[0041]FIG. 13B shows the DNA sequence of human Erβ;

[0042]FIG. 14A shows the amino acid sequence of mERβ;

[0043]FIG. 14B shows the DNA sequence of mouse Erβ; and

[0044]FIG. 15 illustrates ligand binding affinities for variousphytoestrogens by ER's of the invention.

A. CLONING OF RAT ERβ

[0045] 1. PCR-Amplification and Complementary DNA Cloning.

[0046] A set of degenerate primers (DBD 1,2,3 and WAK/FAK) were designedpreviously according to the most highly conserved sequences of theDNA-binding domain (P-box) and ligand binding domain (Ti-stretch) ofmembers of the nuclear receptor family (Enmark, E., Kainu, T.,Pelto-Huikko, M., & Gustafsson, J -Å (1994) Biochem. Biophys. Res.Commun. 204, 49-56). Single strand complementary DNA reverse transcribedfrom rat prostate total RNA was employed with the primers in PCRreactions as described in Enmark, E., Kainu, T., Pelto-Huikko, M., &Gustafsson, J -Å (1994) Biochem. Biophys. Res. Commun. 204, 49-56. Theamplification products were separated on a 2% low melting agarose geland DNA products between 400 and 700 bp were isolated from the gel andligated to TA cloning vector (Invitrogen). As alternatives, we also usedthe RP-I/IRP-2 and DBD66-100/DBD210-238 primer sets in the DNA-bindingdomain of nuclear receptors exactly as described by Hirose T., Fijimoto,W., Yamaai, T., Kim, K. H., Matsuura, H., & Jetten, A. M. (1994) Mol.Endocrinol. 8, 1667-1677 and Chang, C., Lopes Da Silva, S., Ideta, R.,Lee, Y., Yeh, S., & Burbach, J. P. H. (1994) Proc. Natl. Acad. Sci. 91,6040-6044 respectively. Clone number 29 (obtained with the DBD-WAK/FAKset) with a length of 462 bp showed high homology (65%) with the ratestrogen receptor cDNA (65%), which was previously cloned from ratuterus (Koike, S., Sakai, M., & Muramatsu, M. (1987) Nucleic Acids Res15, 2499-2513). The amino acid residues predicted by clone 29 DNAsequences suggested that this DNA fragment encoded part of theDNA-binding domain, hinge region and the beginning of the ligand bindingdomain of a novel member of the nuclear receptor family. Two PCR primers(FIG. 1) were used to generate a probe of 204 bp consisting of the hingeregion of the novel receptor, which was used to screen a rat prostatecDNA library (Clontech gt 10) under stringent conditions resulting infour strongly positive clones with a size of 0.9 kb, 1.8 kb, 2.5 kb and5-6 kb respectively. The clone of 2.5 kb was sequenced and FIG. 1 showsthe nucleotide sequence determined in the core facility (CyberGene AB)by cycle sequencing using fluorescent terminators (Applied Biosystems)on both strands, with a series of internal primers and deduced aminoacid sequence of clone 29. Two in frame ATG codons are located atnucleotide 424 and nucleotide 448, preceding by an in-frame stop codonat nucleotide 319, which suggests that they are possible start codons.The open reading frame encodes a protein of 485 amino acid residues(counted from the first methionine) with a calculated molecular weightof 54.2 kDa. Analysis of the proteins by synthesized by in-vitrotranslation from the clone 29 cRNA in rabbit reticulocyte lysaterevealed a doublet protein band migrating at approximately 57 kDa onSDS-PAGE gels (data not shown), confirming the open reading frame. Thedoublet protein band is probably caused by the use of both ATG codonsfor initiation of protein synthesis. The amino acid sequence of clone 29protein shows the characteristic zinc module DNA-binding domain, hingeregion and a putative ligand binding domain, which are thecharacteristic features of members of the nuclear receptor family (Tsai,M -J., & O'Malley, B. W. (1994) Ann. Rev. Biochem. 63, 451-486; Härd,T., & Gustafsson, J -Å (1993) Acc. Chem. Res. 26, 644-650; Laudet, V.,Hänni, C., Coli, J., Catzeflis, F., & Stehelin, D (1992) EMBL J 11,1003-1012).

[0047] Protein sequence comparison with several representative membersof the nuclear receptor family (FIG. 2) showed the clone 29 protein ismost related to the rat estrogen receptor (ERα), cloned from uterus(Koike, S., Sakai, M., & Muramatsu, M. (1987) Nucleic. Acids Res. 15,2499-2513), with 95% identity in the DNA-binding domain (amino acidresidues 103-167) (Griffiths, K., Davies, P., Eaton, C. I., Harper, M.E., Turkes, A., & Peeling, W. B. (1991) in Endocrine Dependent Tumours,eds. Voigt, K -D. & Knabbe, C. (Raven Press), pp. 83-125). A number offunctional characteristics have been identified within the DNA-bindingdomain of nuclear receptors (Härd, T., & Gustafsson, J -Å. (1993) Acc.Chem. Res 26, 644-650 and Zilliacus, J., Carlstedt-Duke, J., Gustafsson,J -Å., & Wright, A. P. H. (1994) Proc. Natl. Acad. Sci. USA 91,4175-4179). The so-called P-box specifies nucleotide sequencerecognition of the core half-site within the response element, while theD-box mediates dimerization between receptor monomers. The clone 29protein P-box and D-box sequences of EGCKA and PATNQ, respectively, areidentical to the corresponding boxes in ERα (Härd, T., & Gustafsson, J-Å. (1993) Acc. Chem. Res 26, 644-650 and Koike, S., Sakai, M., &Muramatsu, M. (1987) Nucleic Acids Res. 15, 2499-2513), thus predictingthat clone 29 protein binds to ERE sequences.

[0048] The putative ligand binding domain (LBD) of clone 29 protein(amino acid residues 259-457) shows closest homology to the LBD of therat ERα (FIG. 2), while the homology with the human ERR1 and ERR2proteins (Giguere, V., Yang, N., Segui, P., & Evans R. M. (1988) Nature331, 91-94) is considerably less. With the human, mouse and xenopusestrogen receptors the homology in the LBD is also around 55%, while thehomology with the LBD of other steroid receptors is not significant(FIG. 2). Cysteine residue 530 in human ERα has been identified as thecovalent attachment site of an estrogenic affinity label (Harlow, K. W.,Smith D. N., Katzenellenbogen, J. A., Greene, G. L., & Katzenellenbogen,B. S. (1989) J. Biol. Chem. 264, 17476-17485). Interestingly, clone 29protein (Cys-436) as well as the mouse, rat and xenopus ERαs have acysteine residue at the corresponding position. Also, two other aminoacid residues described to be close to or part of the ligand-bindingpocket of the human ERα-LBD (Asp 426 and Gly 521) are conserved in theLBD of clone 29 protein, (Asp 333 and Gly 427) and in the LBD of ERαsfrom various species (20,21). The ligand-dependent transactivationfunction TAF-2 identified in ERα (Danielian, P. S., White, R., Lees, J.A., & Parker, M. G. (1992) EMBO J. 11, 1025-1033), which is believed tobe involved in contacting other transcription factors and therebyinfluencing activation of transcription of tarteg genes, is almostcompletely conserved in clone 29 protein (amino acid residues 441-457).Steroid hormone receptors are phosphoproteins (Kuiper, G., & Brinkmann,A. O. (1994) Mol. Cell. Endocrinol. 100, 103-107), and severalphosphorylation sites identified in the N-terminal domain and LBD of ERα(Arnold, S. F., Oboum, J. D., Jaffe, H., & Notides. A. C. (1995) Mol.Endocrinol. 9, 24-33 and Le Goff, P., Montano, M. M., Schodin, D. J., &Katzenellenbogen, B. S. (1994) J. Biol. Chem. 269, 4458-4466) areconserved in clone 29 protein (Ser 30 and 42, Tyr 443). Clone 29 proteinconsists of 485 amino acid residues while ERαs from human, mouse and ratconsist of 590-600 amino acid residues. The main difference is a muchshorter N-terminal domain in clone 29 protein i.e 103 amino acidresidues as compared to 185-190 amino acid residues in the otherreceptor proteins. Also the non-conserved so-called F-domain at theC-terminal end of ERαs is 15 amino acid residues shorter in clone 29protein. The cDNA insert of a positive clone of 2.6 kb was subclonedinto the EcoR1 site of pBluescript (trademark) (Stratagene). Thecomplete DNA sequence of clone 29 was determined (CyberGene AB) by cyclesequencing using fluorescent terminators (Applied Biosystems) on bothstrands, with a series of internal primers.

[0049]FIGS. 2C and 2D respectively compare the ligand and DNA bindingdomain of Erβ compared to rat, mouse and human Erα's .

[0050] 2. Saturation Ligand Binding Analysis and Ligand CompetitionStudies:

[0051] Clone 29 cDNA was subcloned in pBluescript downstream of the T7promoter to give p29-T7. Clone 29 protein was synthesized in vitro usingthe TnT-coupled reticulocyte lysate system (Promega). Translationreaction mixtures were diluted five times with TEDGMo buffer (40 mmTris/HCl, pH 7.4, 1mM EDTA, 10% (v/v) glycerol, 10 mM Na₂MoO₄, 10 mMDTT) and 0.1 ml aliquots were incubated for 16 h at 8° C. with 0.3-6.2nM [2,4,6,7-³H]-17β-estradiol (NEN-Dupont; specific radioactivity 85Ci/mmol) in the presence or absence of a 200-fold excess of unlabelledE2.

[0052]FIG. 5A illustrates the results of a saturation ligand analysis ofclone 29 protein. Reticulocyte lysate containing clone 29 protein wasincubated with 6 concentrations of [³H]E2 between 0.3 and 6.0 nM.Parallel tubes contained an additional 200 fold of non-radioactive E2.Bound and free ligand were separated with a dextran-coated charcoalassay. The Kd (0.6 nM) was calculated from the slope of the line in theScatchard plot shown (r=0.93), and the number of binding sites wasextrapolated from the intercept on the abscissa (Bmax=1400 fmol/mlundiluted translation mixture).

[0053] For ligand competition studies diluted reticulocyte lysate wasincubated with 5 nM [2,4,6,7-³H]-17β-estradiol in the presence of either0, 5, 50, 500 or 5,000 nM of non-radioactive E2, estrone, estriol,testosterone, progesterone, corticosterone, 5α-androstane-3β,17β-diol,5α-androstanc-3α,17β,-diol and diethylstilbestrol (DCES) for 16 h at 8°C. Bound and unbound steroids were separated with a dextran-coatedcharcoal assay (Ekman, P., Barrack, E. R., Greene, G. L., Jensen, E. V.,& Walsh, P. C. (1983) J. Clin. Endocrinol Metab. 57, 166-176).

[0054]FIG. 5B illustrates the specificity of ligand binding by clone 29protein. Reticulocyte lysate containing clone 29 protein wasequilibrated for 16 h with 5 nM [³H]E2 and the indicated fold excess ofcompetitors. Data represent [³H]E2 bound in the presence of unlabelledE2, testosterone (T), progesterone (prog), corticosterone (cortico),estrone (E1), diethylstilbestrol (DES), 5α-androstane-3α, 17β-diol(3α-AD), 5α-androstane- 3β, 17β-diol (3β-AD) and estriol (E3). [³H]E2binding in the absence of competitor was set at 100%.

[0055] 3. In-Situ Hybridisation:

[0056] In-situ hybridisation was carried out as previously described(Dagerlind Å., Friberg, K., Bean, A. J., & Hökfelt, T (1992)Histochemistry 98, 39-49). Briefly, two oligonucleotide probes directedagainst nucleotides 994-1041 and 1981-2031 were each labelled at the3′-end with-³³P-dATP using terminal deoxynucleotidyltransferase(Amersham, UK). Adult male and female Sprague-Dawley rats (age 2 to 3months n=10) were used for this study. The rats were decapitated and thetissues were rapidly excised and frozen on dry ice. The tissues weresectioned in a Microm HM500 cryostat at 14 μm and thawed onto Probe-Onglass slides (Fisher Scientific, PA, USA). The slides were stored at−20° C. until used. The slides were incubated in humidifed boxes at 42°C. for 18 h with 1×10⁶ cpm of the probe in a hybridization solutioncontaining 50% formamide, 4×SSC (1×SSC=0.15 M NaCl, 0.015 M sodiumcitrate), 1×Denhardt (0.02% BSA, 0.02% Ficoll, 0.02% PVP), 1% sarkosyl,0.02 M sodium phosphate (pH 7.), 10% dextransulphate, 500 μg/ml salmonsperm DNA and 200 mM DTT. Slides were subsequently rinsed in 1×SSC at55° C. for 60 min with four changes of SSC and finally in 1×SSC startingat 55° C. and slowly cooled to room temperature, transferred throughdistilled water and briefly dehydrated in 50% and 95% ethanol for 30 seceach, air-dried, and covered with Amersham β-man autoradiography filmfor 15 to 30 days. Alternatively the slides were dipped in Kodak NTB2nuclear track emulsion (diluted 1:1 with distilled water) and exposedfor 30 to 60 days at 4° C. Finally, the sections were stained withcresyl violet.

[0057] Clear expression of clone 29 was observed in the reproductivetract of both male and female rats, while in all other rat tissues theexpression was very low or below the level of detection with in-situhybridisation (not shown). In male reproductive organs high expressionwas seen in the prostate gland (FIG. 3), while very low expression wasobserved in testis, epididymis and vesicula seminalis (not shown). Indipped sections, expression was clearly visible in prostate epithelialcells (secreting alveoli) while the expression in smooth muscle cellsand fibroblasts in the stroma was low (FIG. 3). In female reproductiveorgans expression was seen in the ovary (FIG. 4), while uterus andvagina were negative (not shown). In dipped sections high expression wasseen in the granulosa cell layer of primary, secondary and maturefollicles (FIG. 4), whereas primordial follicles, oocytes and corporalutea appeared completely negative. Low expression was seen in theinterstitial cells of the ovary. Both anti-sense oligonucleotide probesused produced similar results. Addition of a 100 fold excess of therespective unlabelled oligonucleotide probes during the hybridisationreactions abolished all signals.

[0058] 4. Transactivation Analysis in CHO-Cells:

[0059] The expression vector pCMV29 was constructed by inserting the 2.6kb clone 29 fragment in the EcoRI site of the expression vector pCMV5(Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., & Russell, D.W. (1989) J. Biol. Chem. 264, 8222-8229). The pERE-ALP reporterconstruct contains a secreted form of the plancental alkalinephosphatase gene (Berger, J., Hauber, J., Hauber, R., Geiger, R., &Cullen, B. R. (1988) Gene 66, 1-10) and the MMTV-LTR in which theglucocorticoid response elements were replaced by the vitellogeninpromoter estrogen response element (ERE).

[0060] CHO—K1 cells were seeded in 12-well plates at approximately1.7×10⁵ cells per well in phenol-red free Ham F12 medium with 5% FCS(dextran-coated charcoal treated) and 2 mM Lglutamine. After 24 h thecells were transfected with 250 ng pERE-ALP vector and 50 ng pCMV29using lipofectamine (Gibco) according to the manufacturer'sinstructions. After five hours of incubation the cells were washed andrefed with 0.5 ml phenol-red free Coon's F-12 medium containing 5% serumsubstitute (SRC 3000, Tissue Culture Services Ltd., Botolph Claydon,Buckingham, UK) 2 mM Lglutamine and 50 μg/ml gentamicin plus hormones asindicated. After 48 h the medium was assayed for alkaline phosphatase(ALP) activity by a chemiluminescence assay. A 10 μl aliquot of the cellculture medium was mixed with 200 μl assay buffer (10 mM diethanolaminepH 10.0 1 mM MgCl₂ and 0.5 mM CSPD (Tropix Inc. Boston, USA) ) andincubated for 20 min at 37° C. before measurement in a microplateluminometer (Luminoskan; Labsystems, Finland) with integral measurementfor 1 second. The ALP activity of ERE-reporter alone was set at 1.

[0061] 5. Ligand Binding Characteristics and Transactivation Function ofClone 29 Protein:

[0062] On the basis of the described high homology between clone 29protein and rat ERα in the DBD and LBD it was hypothesized that clone 29protein might encode a novel ER. Furthermore, biological effects ofestrogens on rat prostate and ovary, which show high expression of clone29 RNA, are well known (Griffiths, K., Davies, P., Eaton, C. I., Harper,M. E., Turkes, A., & Peeling W. B. (1991) in Endocrine DependentTumours, eds Voigt, K -D. & Knabbe, C. (Raven Press), pp 83-125;Richards, J. S (1994) Endocrine Rev. 15, 725-745; and Habenicht, U -F.,Tunn, U. W., Senge, Th., Schroder, R. H., Schweikert, H. U., Bartsch,G., & El Etreby, M. F. (1993) J. Steroid Biochem. Molec. Biol. 44,557-563). In order to analyze the steroid binding properties of clone 29protein synthesized in vitro, the reticulocyte lysate was incubated at8° C. for 16 h with increasing concentrations (0.3-6.0 nM) of [³H]E2 inthe presence or absence of a 200 fold molar excess of unlabelled E2.Linear transformation of saturation data-revealed a single population ofbinding sites for E2 with a K_(d) (dissociation constant) of 0.6 nM(FIGS. 5A and C). Steroid binding specificity was measured by incubatingreticulocyte lysate with 5 nM [³H]E2 in the presence of 0.5, 50, 500 and5,000 nM unlabelled competitors. Competition curves generated areindicative of an estrogen receptor in that only estrogens competedefficiently with [³H]E2 for binding (FIG. 5B). Fifty percent inhibitionof specific binding occured by 0.6 fold excess of unlabelled E2;diethylstilbestrol, estriol, estrone and 5α-androstane-3α,17β-diol were5, 15, 50 and 150 times, respectively, less effective as competitors.Neither testosterone, progesterone, corticosterone nor5α-androstane-3α,17β-diol were efficient competitors, even at thehighest concentrations used (1000 fold excess). The dissociationconstant and the steroid binding specificities measured are in goodagreement with data previously reported for ERs in rat and humanprostate, rat granulosa cells, rat antral follicles and whole ratovarian tissue (Ekman, P., Barrack, E. R., Greene, G. L., Jensen, E. V.,& Walsh. P. C (1983) J. Clin. Endocrinol. Metab. 57, 166-176; vanBeurden-Lamers, W. M. O., Brinkmann, A. O., Mulder, E., & van der Molen,H. (1974) Biochem. J 140, 495-502; Kudolo, G. B., Elder, M. G., & Myatt,L. (1984) J. Endocrinol. 102, 83-91; and Kawashima, M., & Greenwald, G.S. (1993) Biology of Reprod. 48 172-179).

[0063] When clone 29 protein was labelled with a saturating dose of[³H]E2 and analyzed on sucrose density gradients, a single peak ofspecifically bound radioactivity was observed. The sedimentationcoefficient of this complex was about 7S, and it shifted to 4S in thepresence of 0.4 M NaCl (not shown). To investigate the transcriptionalregulatory properties of clone 29 protein, we performed co-transfectionexperiments in which CHO cells were transfected with a clone 29 proteinexpression vector and/or an estrogen-responsive reporter gene construct.Cells were incubated in the absence of E2 (clone 29) or in the presenceof 100 nM E2 (Clone 29+E2) or in the presence of 100 nM E2 and 12 μMTamoxifen (Clone 29+E2/Tam). In the absence of exogenously added E2clone 29 protein showed considerable transcriptional activity whichcould be further increased by the addition of 100 nM E2 (FIG. 6).Simultaneous addition of a 10 fold excess of the antiestrogen Tamoxifenpartially suppressed the E2 stimulated activity (FIG. 6). Theconstitutive transcriptional activity of clone 29 protein could besuppressed by the anti-estrogen ICI-1624384 (not shown). It has beenshown previously that the wild-type mouse and human ERs are constitutiveactivators of transcription, and that the transcriptional activity canbe stimulated further by the addition of E2 (Txukernan, M., Xiao-KunZhang., Hermann, T., Wills, K. N., Graupner, G., & Phal, M. (1990) NewBiologist 2, 613-620 and Lees, J. A., Fawell, S. E., & Parker, M. G.(1989) Nucl. Acids Res. 17, 5477-5488). To obtain more insight into whatconcentrations of E2 effect clone 29 protein transcriptional activity,transient transfection experiments were carried out in the presence ofincreasing concentrations of E2. CHO-cells were transiently transfectedwith the ERE-reporter plasmid and the clone 29 protein expressionplasmid. Cells were incubated with increasing concentrations of E2 (0.1-1000 nM), estrone (E1, 1000 nM), 5α-androstane-3β,17β-diol (3β-AD, 1000nM) or no ligand added. Alkaline phosphatase activity (ALP) was measuredas described and the activity in the absence of ligand (control) was setat 1. The figure shows relative ALP-activities (±SD) from threeindependent experiments. Clone 29 protein began to respond at 0.1 nM E2and maximal stimulation was observed between 1 nm and 10 nM E2 (FIG. 7).The maximal stimulation factor was 2.6±0.5 fold (mean±SD. n=9) ascompared to incubation in the absence of E2. Apart from E2 also estroneand 5α-androstane- 3β,17β-diol could stimulate transcriptional activity,albeit at higher concentrations (FIG. 7). Dexamethasone, testosterone,progesterone, 5α-androstane-3α,17β-diol, thyroid hormone andall-trans-retinoic acid could not stimulate transcriptional activity ofclone 29 protein, even at the highest concentration (1000 nM) tested(not shown). The results of the co-transfection experiments are inagreement with the ligand binding and specificity data of clone 29protein presented in FIG. 5. In control experiments, wild-type human ERαalso showed transcriptional activity in the absence of E2, which couldbe increased by the addition of E2 (not shown).

[0064] 6. Detection of Rat ER Expression by RT-PCR

[0065] The tissue specificity of expression of rat ERβ and ERα wasdetermined using reverse transcriptase polymerase chain reaction(RT-PCR). The results of the experiment are shown in FIG. 8.

[0066] B. Isolation of Human Erβ

[0067] 1. A human version of Erβ (hERβ) has also been cloned from humanovary. The tissue specificity of hERβ expression in a variety of cellswas also determined using the RT-PCR technique. The results are shown inFIG. 9. It will be noticed that there is a very high level of mRNA ofhERβ in human umbilical vein endothelial cells (HUVEC) but no detectionof hERα in the same cells. In addition, it will be seen that in humanosteosarcoma cell line (HOS-D4), hERβ is expressed in greater quantitiescompared to hERα.

[0068] I. A human version of ERβ (hERβ) has also been cloned. The tissuespecificity of hERβ expression in a variety of cells was also determinedusing the RT-PCR technique. The results are shown in FIG. 9. It will benoticed that there is a very high level of mRNA of hERβ in humanumbilical vein endothelial cells (HUVEC) but no detection of hERα in thesame cells. In addition, it will be seen that in human osteosarcoma cellline (HOS-D4), hERβ is expressed in greater quantities compared to hERα.

[0069] The partial DNA sequence of hERβ is shown in FIG. 10 and aderived amino acid sequence is shown in FIG. 11.

[0070] Cloning of Human Erβ from Testis

[0071] A commercially available cDNA from human testis (Clontech,article no. HL1161x) was screened, using a fragment containing theligand-binding domain of the rat Erβ cDNA as probe. Approximately 10⁶recombinants were screened, resulting in one positive clone. Uponsequencing of this clone, it was seen that the insert was 1156 bp (FIGS.13A and 13B). This corresponds to most of the translated region of areceptor with an overall homology of 90.0% to rat Erβ, therefore deducedto represent the human form of Erβ.

[0072] The cloned hERβ, however, lacks approximately 47 amino acids atthe N-terminal end and 61 amino acids at the C-terminal end (as comparedto the rat sequence). Further screening of the same library wasunsuccessful. PCR technology was therefore used to obtain the remainingparts. For oligonucleotides were sunthesised; two degenerateoligonucleotides containing all possible codons for the amino acidsadjacent to the initiation methionine and the stop codon, respectively,of the rat Erβ, and two specific oligonucleotides containing thesequence of the clone isolated from the human testis library andsituated approximately 100 bp from respective end of this clone. PCRwith the N-terminal and C-terminal pair of oligos yielded specificbands, that were subcloned and sequenced. The parts of these new clonesthat overlap the original cDNA clone are identical to this. It was thuspossible to construct peptide and DNA sequences corresponding to thewhole open reading frame (FIGS. 13A and 13B).

[0073] When comparing the human Erβ to rat Erβ, this receptor is 79.6%identical in the N-terminal domain, 98.5% in the DNA-binding domain,85.6% in the hinge and 91.6% in the ligand-binding and F-domains. Thesenumbers match very well those found when comparing the rat and humanforms of Erα.

[0074] Studies of the expression of human Erβ using Northern blot showexpression in testis and in ovaries. The expression in prostate,however, appears lower than found in the rat.

[0075] The human Erβ gene has been mapped to chromosome 14 using PCR andto region 14q22-23 using the FISH technique, whereas the human Erβ genehas been mapped to chromosome 6q25.

[0076] 2. Comparison of Ligand Binding Affinity of hERα and rERβ

[0077] The ligand affinity of the two estrogen receptors, human Erα(ovary) (hERβ) and rat Erβ (rERβ) was tested in binding saturationexperiments and in binding competition experiments.

[0078] cDNA of the receptor subtypes hERα and rERβ were in vitrotranslated in rabbit reticulocyte lysate in presence of non-radioactiveamino acids according to the instructions supplied by the manufacturer(Promega).

[0079] The radioactive ligand used in all experiments was16α[¹²⁵I]-17β-estradiol ([¹²⁵I]-E2) (NEX-144, New England Nuclear). Themethod for the binding experiments was previously described in:Salomonsson M, Carlsson B, Haggblad J. J. Steroid Biochem. Molec. Biol.Vol. 50, No. 5/6 pp. 313-18, 1994. In brief, estrogen receptors areincubated with [¹²⁵I]-E2 to equilibrium (16-18 h at +4° C.). Theincubation was stopped by separation of protein-bound [¹²⁵I]-E2 fromfree [¹²⁵I]-E2 on Sephadex G25 columns. The radioactivity of the eluateis measured in a gamma-counter.

[0080] In the competition experiments, non-radioactive ligands werediluted in DMSO, mixed with [¹²⁵I]-E2 (approximately 100-200 pM),aliquoted in parallel, and finally hERα or rERβ was added. The finalconcentration of DMSO in the binding buffer was 2%.

[0081] The buffer used in the experiments was of the followingcomposition: Hepes (pH=7.5) 20 mM, KCl 150 mM, EDTA 1 mM, glycerol(8.7%), monothioglycerol 6 mM, Na₃MO₄10 mM.

[0082] 3. Equilibrium Binding Saturation Experiments(K_(d)-Determinations)

[0083] A range of concentrations of [¹²⁵I]-E2 were mixed with the ER:sand incubated as described above, free [¹²⁵I]-E2 was determined bysubstracting bound [¹²⁵I]-E2 from added [¹²⁵I]-E2. Binding data wasanalysed by Hill-plots and by Scatchard plots (FIG. 11). The equilibriumbinding results are shown in Table 1. The apparent K_(d)-values for[¹²⁵I]-E2 differed between the two ER:s with approximately a factor offour; K_(d)(hERα):K_(d)(rERβ)=1:4. TABLE 1 Equilibrium dissociationconstants for [¹²⁵I]-E2 to the two subtypes. Receptor subtype K_(d)(Hill-plot) K_(d) (Scatchard-plot) hERα 0.06 nM 0.09 nM eERβ 0.24 nM0.42 nM

[0084] 4. Competition Experiments (IC₅₀ Determinations)

[0085] The experiments were performed as described above. IC₅₀ valueswere obtained by applying a four parameter logistic analysis;b=((b_(max)−b_(min))/(1+(I/IC₅₀)^(S)))+b_(min), where I is the addedconcentration of binding inhibitor, IC₅₀ is the concentration ofinhibitor at half maximal binding and S is a slope factor. The freeconcentration of [¹²⁵I]-E2 was determined by sampling an aliquot fromthe wells at the end of the incubation and then substract boundradioactivity from sampled total radioactivity.

[0086] Since the equilibrium binding experiments (above) showed that theK_(d)-values for [¹²⁵I]-E2 differed between the two ER:s, K₁-values(from the Cheng-Prusoff equation: K₁=IC₅₀/(1+L/K_(d)) where L is free([¹²⁵I]-E2]) were calculated for the compounds investigated. Twoapproaches for calculating RBA (Relative Binding Affinity) were used.The RBA values were derived using either the IC₅₀ values or the K₁values. In both approaches, the value for the compound16α-bromo-estradiol was selected as the reference value (100%). Bothapproaches gave similar results. The results are summarized in FIG. 12.In these Figures “4-OH-Tam”=4-hydroxy-tamoxifen;“DES”=diethylstilbestrol; “Hexestr”=hexestrol; “ICI-164384”=ICI plccompound no. 164382; “17β-E2”=17β-estradiol;“16a-B-E2”=16α-bromo-estradiol; “Ralox”=Raloxifen; and “17a-E2”=17αdiol.

[0087] The results show that Erα and Erβ have significant differentligand binding affinities—the apparent K_(d)-values for [¹²⁵I]-E2differed between the two ER's by a factor of about 4 (K_(d)(hERα):K_(d)(rERβ)≈1:4). Some compounds investigated showed significantdifferences in the competition for binding of [¹²⁵I]-E2 to the ER's.Certain compounds were found to be more potent inhibitors of [¹²⁵I]-E2binding to hERα as compared to rERβ whereas others were found to be morepotent inhibitors of [¹²⁵I]-E2 binding to rERβ than to hERα.

1 19 2568 base pairs nucleic acid double linear Rattus rattus 1GGAATTCCGG GGGAGCTGGC CCAGGGGGAG CGGCTGGTGC TGCCACTGGC ATCCCTAGGC 60ACCCAGGTCT GCAATAAAGT CTGGCAGCCA CTGCATGGCT GAGCGACAAC CAGTGGCTGG 120GAGTCCGGCT CTGTGGCTGA GGAAAGCACC TGTCTGCATT TAGAGAATGC AAAATAGAGA 180ATGTTTACCT GCCAGTCATT ACATCTGAGT CCCATGAGTC TCTGAGAACA TAATGTCCAT 240CTGTACCTCT TCTCACAAGG AGTTTTCTCA GCTGCGACCC TCTGAAGACA TGGAGATCAA 300AAACTCACCG TCGAGCCTTA GTTCCCTGCT TCCTATAACT GTAGCCAGTC CATCCTACCC 360CTGGAGCACG GCCCCATCTA CATCCCTTCC TCCTACGTAG ACAACCGCCA TGAGTATTCA 420GCTATGACAT TCTACAGTCC TGCTGTGATG AACTACAGTG TTCCCGGCAG CACCAGTAAC 480CTGGACGGTG GGCCTGTCCG ACTGAGCACA AGCCCAAATG TGCTATGGCC AACTTCTGGG 540CACCTGTCTC CTTTAGCGAC CCATTGCCAA TCATCGCTCC TCTATGCAGA ACCTCAAAAG 600AGTCCTTGGT GTGAAGCAAG ATCACTAGAG CACACCTTAC CTGTAAACAG AGAGACACTG 660AAGAGGAAGC TTAGTGGGAG CAGTTGTGCC AGCCCTGTTA CTAGTCCAAA CGCAAAGAGG 720GATGCTCACT TCTGCCCCGT CTGCAGCGAT TATGCATCTG GGTATCATTA CGGCGTTTGG 780TCATGTGAAG GATGTAAGGC CTTTTTTAAA AGAAGCATTC AAGGACATAA TGATTATATC 840TGTCCAGCCA CGAATCAGTG TACCATAGAC AAGAACCGGC GTAAAAGCTG CCAGGCCTGC 900CGACTTCGCA AGTGTTATGA AGTAGGAATG GTCAAGTGTG GATCCAGGAG AGAACGGTGT 960GGGTACCGTA TAGTGCGGAG GCAGAGAAGT TCTAGCGAGC AGGTACACTG CCTGAGCAAA 1020GCCAAGAGAA ACGGTGGGCA TGCACCCCGG GTGAAGGAGC TACTGCTGAG CACCTTGAGT 1080CCAGAGCAAC TGGTGCTCAC CCTCCTGGAA GCTGAACCAC CCAATGTGCT GGTGAGCCGT 1140CCCAGCATGC CCTTCACCGA GGCCTCCATG ATGATGTCCC TCACTAAGCT GGCGGACAAG 1200GAACTGGTGC ACATGATTGG CTGGGCCAAG AAAATCCCTG GCTTTGTGGA GCTCAGCCTG 1260TTGGACCAAG TCCGGCTCTT AGAAAGCTGC TGGATGGAGG TGCTAATGGT GGGACTGATG 1320TGGCGCTCCA TCGACCACCC CGGCAAGCTC ATTTTCGCTC CCGACCTCGT TCTGGACAGG 1380GATGAGGGGA AGTGCGTAGA AGGGATTCTG GAAATCTTTG ACATGCTCCT GGCGACGACG 1440TCAAGGTTCC GTGAGTTAAA ACTCCAGCAC AAGGAGTATC TCTGTGTGAA GGCCATGATC 1500CTCCTCAACT CCAGTATGTA CCCCTTGGCT TCTGCAAACC AGGAGGCAGA AAGTAGCCGG 1560AAGCTGACAC ACCTACTGAA CGCGGTGACA GATGCCCTGG TCTGGGTGAT TGCGAAGAGT 1620GGTATCTCCT CCCAGCAGCA GTCAGTCCGA CTGGCCAACC TCCTGATGCT TCTTTCTCAC 1680GTCAGGCACA TCAGTAACAA GGGCATGGAA CATCTGCTCA GCATGAAGTG CAAAAATGTG 1740GTCCCGGTGT ATGACCTGCT GCTGGAGATG CTGAATGCTC ACACGCTTCG AGGGTACAAG 1800TCCTCAATCT CGGGGTCTGA GTGCAGCTCA ACAGAGGACA GTAAGAACAA AGAGAGCTCC 1860CAGAACCTAC AGTCTCAGTG ATGGCCAGGC CTGAGGCGGA CAGACTACAG AGATGGTCAA 1920AAGTGGAACA TGTACCCTAG CATCTGGGGG TTCCTCTTAG GGCTGCCTTG GTTACGCACC 1980CCTTACCCAC ACTGCACTTC CCAGGAGTCA GGGTGGTTGT GTGGCGGTGT TCCTCATACC 2040AGGATGTACC ACCGAATGCC AAGTTCTAAC TTGTATAGCC TTGAAGGCTC TCGGTGTACT 2100TACTTTCTGT CTCCTTGCCC ACTTGGAAAC ATCTGAAAGG TTCTGGAACT AAAGGTCAAA 2160GTCTGATTTG GAAGGATTGT CCTTAGTCAG GAAAAGGAAT ATGGCATGTG ACACAGCTAT 2220AAGAAATGGA CTGTAGGACT GTGTGGCCAT AAAATCAACC TTTGGATGGC GTCTTCTAGA 2280CCACTTGATT GTAGGATTGA AAACCACATT GACAATCAGC TCATTTCGCA TTCCTGCCTC 2340ACGGGTCTGT GAGGACTCAT TAATGTCATG GGTTATTCTA TCAAAGACCA GAAAGATAGT 2400GCAAGCTTAG ATGTACCTTG TTCCTCCTCC CAGACCCTTG GGTTACATCC TTAGAGCCTG 2460CTTATTTGGT CTGTCTGAAT GTGGTCATTG TCATGGGTTA AGATTTAAAT CTCTTTGTAA 2520TATTGGCTTC CTTGAAGCTA TGTCATCTTT CTCTCTCTCC CGGAATTC 2568 485 aminoacids amino acid linear Rattus rattus 2 Met Thr Phe Tyr Ser Pro Ala ValMet Asn Tyr Ser Val Pro Gly Ser 1 5 10 15 Thr Ser Asn Leu Asp Gly GlyPro Val Arg Leu Ser Thr Ser Pro Asn 20 25 30 Val Leu Trp Pro Thr Ser GlyHis Leu Ser Pro Leu Ala Thr His Cys 35 40 45 Gln Ser Ser Leu Leu Tyr AlaGlu Pro Gln Lys Ser Pro Trp Cys Glu 50 55 60 Ala Arg Ser Leu Glu His ThrLeu Pro Val Asn Arg Glu Thr Leu Lys 65 70 75 80 Arg Lys Leu Ser Gly SerSer Cys Ala Ser Pro Val Thr Ser Pro Asn 85 90 95 Ala Lys Arg Asp Ala HisPhe Cys Pro Val Cys Ser Asp Tyr Ala Ser 100 105 110 Gly Tyr His Tyr GlyVal Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe 115 120 125 Lys Arg Ser IleGln Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn 130 135 140 Gln Cys ThrIle Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg 145 150 155 160 LeuArg Lys Cys Tyr Glu Val Gly Met Val Lys Cys Gly Ser Arg Arg 165 170 175Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Gln Arg Ser Ser Ser Glu 180 185190 Gln Val His Cys Leu Ser Lys Ala Lys Arg Asn Gly Gly His Ala Pro 195200 205 Arg Val Lys Glu Leu Leu Leu Ser Thr Leu Ser Pro Glu Gln Leu Val210 215 220 Leu Thr Leu Leu Glu Ala Glu Pro Pro Asn Val Leu Val Ser ArgPro 225 230 235 240 Ser Met Pro Phe Thr Glu Ala Ser Met Met Met Ser LeuThr Lys Leu 245 250 255 Ala Asp Lys Glu Leu Val His Met Ile Gly Trp AlaLys Lys Ile Pro 260 265 270 Gly Phe Val Glu Leu Ser Leu Leu Asp Gln ValArg Leu Leu Glu Ser 275 280 285 Cys Trp Met Glu Val Leu Met Val Gly LeuMet Trp Arg Ser Ile Asp 290 295 300 His Pro Gly Lys Leu Ile Phe Ala ProAsp Leu Val Leu Asp Arg Asp 305 310 315 320 Glu Gly Lys Cys Val Glu GlyIle Leu Glu Ile Phe Asp Met Leu Leu 325 330 335 Ala Thr Thr Ser Arg PheArg Glu Leu Lys Leu Gln His Lys Glu Tyr 340 345 350 Leu Cys Val Lys AlaMet Ile Leu Leu Asn Ser Ser Met Tyr Pro Leu 355 360 365 Ala Ser Ala AsnGln Glu Ala Glu Ser Ser Arg Lys Leu Thr His Leu 370 375 380 Leu Asn AlaVal Thr Asp Ala Leu Val Trp Val Ile Ala Lys Ser Gly 385 390 395 400 IleSer Ser Gln Gln Gln Ser Val Arg Leu Ala Asn Leu Leu Met Leu 405 410 415Leu Ser His Val Arg His Ile Ser Asn Lys Gly Met Glu His Leu Leu 420 425430 Ser Met Lys Cys Lys Asn Val Val Pro Val Tyr Asp Leu Leu Leu Glu 435440 445 Met Leu Asn Ala His Thr Leu Arg Gly Tyr Lys Ser Ser Ile Ser Gly450 455 460 Ser Glu Cys Ser Ser Thr Glu Asp Ser Lys Asn Lys Glu Ser SerGln 465 470 475 480 Asn Leu Gln Ser Gln 485 485 amino acids amino acidlinear Homo sapiens 3 Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr SerIle Pro Ser Asn 1 5 10 15 Val Thr Asn Leu Glu Gly Gly Pro Gly Arg GlnThr Thr Ser Pro Asn 20 25 30 Val Leu Trp Pro Thr Pro Gly His Leu Ser ProLeu Val Val His Arg 35 40 45 Gln Leu Ser His Leu Tyr Ala Glu Pro Gln LysSer Pro Trp Cys Glu 50 55 60 Ala Arg Ser Leu Glu His Thr Leu Pro Val AsnArg Glu Thr Leu Lys 65 70 75 80 Arg Lys Val Ser Gly Asn Arg Cys Ala SerPro Val Thr Gly Pro Gly 85 90 95 Ser Lys Arg Asp Ala His Phe Cys Ala ValCys Ser Asp Tyr Ala Ser 100 105 110 Gly Tyr His Tyr Gly Val Trp Ser CysGlu Gly Cys Lys Ala Phe Phe 115 120 125 Lys Arg Ser Ile Gln Gly His AsnAsp Tyr Ile Cys Pro Ala Thr Asn 130 135 140 Gln Cys Thr Ile Asp Lys AsnArg Arg Lys Ser Cys Gln Ala Cys Arg 145 150 155 160 Leu Arg Lys Cys TyrGlu Val Gly Met Val Lys Cys Gly Ser Arg Arg 165 170 175 Glu Arg Cys GlyTyr Arg Leu Val Arg Arg Gln Arg Ser Ala Asp Glu 180 185 190 Gln Leu HisCys Ala Gly Lys Ala Lys Arg Ser Gly Gly His Ala Pro 195 200 205 Arg ValArg Glu Leu Leu Leu Asp Ala Leu Ser Pro Glu Gln Leu Val 210 215 220 LeuThr Leu Leu Glu Ala Glu Pro Pro His Val Leu Ile Ser Arg Pro 225 230 235240 Ser Ala Pro Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu 245250 255 Ala Asp Lys Glu Leu Val His Met Ile Ser Trp Ala Lys Lys Ile Pro260 265 270 Gly Phe Val Glu Leu Ser Leu Phe Asp Gln Val Arg Leu Leu GluSer 275 280 285 Cys Trp Met Glu Val Leu Met Met Gly Leu Met Trp Arg SerIle Asp 290 295 300 His Pro Gly Lys Leu Ile Phe Ala Pro Asp Leu Val LeuAsp Arg Asp 305 310 315 320 Glu Gly Lys Cys Val Glu Gly Ile Leu Glu IlePhe Asp Met Leu Leu 325 330 335 Ala Thr Thr Ser Arg Phe Arg Glu Leu LysLeu Gln His Lys Glu Tyr 340 345 350 Leu Cys Val Lys Ala Met Ile Leu LeuAsn Ser Ser Met Tyr Pro Leu 355 360 365 Val Thr Ala Thr Gln Asp Ala AspSer Ser Arg Lys Leu Ala His Leu 370 375 380 Leu Asn Ala Val Thr Asp AlaLeu Val Trp Val Ile Ala Lys Ser Gly 385 390 395 400 Ile Ser Ser Gln GlnGln Ser Met Arg Leu Ala Asn Leu Leu Met Leu 405 410 415 Leu Ser His ValArg His Ala Ser Asn Lys Gly Met Glu His Leu Leu 420 425 430 Asn Met LysCys Lys Asn Val Val Pro Val Tyr Asp Leu Leu Leu Glu 435 440 445 Met LeuAsn Ala His Val Leu Arg Gly Cys Lys Ser Ser Ile Thr Gly 450 455 460 SerGlu Cys Ser Pro Ala Glu Asp Ser Lys Ser Lys Glu Gly Ser Gln 465 470 475480 Asn Leu Gln Ser Gln 485 1460 base pairs nucleic acid double linearHomo sapiens 4 CTATGACATT CTACAGTCCT GCTGTGATGA ATTACAGCAT TCCCAGCAATGTCACTAACT 60 TGGAAGGTGG GCCTGGTCGG CAGACCACAA GCCCAAATGT GTTGTGGCCAACACCTGGGC 120 ACCTTTCTCC TTTAGTGGTC CATCGCCAGT TATCACATCT GTATGCGGAACCTCAAAAGA 180 GTCCCTGGTG TGAAGCAAGA TCGCTAGAAC ACACCTTACC TGTAAACAGAGAGACACTGA 240 AAAGGAAGGT TAGTGGGAAC CGTTGCGCCA GCCCTGTTAC TGGTCCAGGTTCAAAGAGGG 300 ATGCTCACTT CTGCGCTGTC TGCAGCGATT ACGCATCGGG ATATCACTATGGAGTCTGGT 360 CGTGTGAAGG ATGTAAGGCC TTTTTTAAAA GAAGCATTCA AGGACATAATGATTATATTT 420 GTCCAGCTAC AAATCAGTGT ACAATCGATA AAAACCGGCG CAAGAGCTGCCAGGCCTGCC 480 GACTTCGGAA GTGTTACGAA GTGGGAATGG TGAAGTGTGG CTCCCGGAGAGAGAGATGTG 540 GGTACCGCCT TGTGCGGAGA CAGAGAAGTG CCGACGAGCA GCTGCACTGTGCCGGCAAGG 600 CCAAGAGAAG TGGCGGCCAC GCGCCCCGAG TGCGGGAGCT GCTGCTGGACGCCCTGAGCC 660 CCGAGCAGCT AGTGCTCACC CTCCTGGAGG CTGAGCCGCC CCATGTGCTGATCAGCCGCC 720 CCAGTGCGCC CTTCACCGAG GCCTCCATGA TGATGTCCCT GACCAAGTTGGCCGACAAGG 780 AGTTGGTACA CATGATCAGC TGGGCCAAGA AGATTCCCGG CTTTGTGGAGCTCAGCCTGT 840 TCGACCAAGT GCGGCTCTTG GAGAGCTGTT GGATGGAGGT GTTAATGATGGGGCTGATGT 900 GGCGCTCAAT TGACCACCCC GGCAAGCTCA TCTTTGCTCC AGATCTTGTTCTGGACAGGG 960 ATGAGGGGAA ATGCGTAGAA GGAATTCTGG AAATCTTTGA CATGCTCCTGGCAACTACTT 1020 CAAGGTTTCG AGAGTTAAAA CTCCAACACA AAGAATATCT CTGTGTCAAGGCCATGATCC 1080 TGCTCAATTC CAGTATGTAC CCTCTGGTCA CAGCGACCCA GGATGCTGACAGCAGCCGGA 1140 AGCTGGCTCA CTTGCTGAAC GCCGTGACCG ATGCTTTGGT TTGGGTGATTGCCAAGAGCG 1200 GCATCTCCTC CCAGCAGCAA TCCATGCGCC TGGCTAACCT CCTGATGCTCCTGTCCCACG 1260 TCAGGCATGC GAGTAACAAG GGCATGGAAC ATCTGCTCAA CATGAAGTGCAAAAATGTGG 1320 TCCCAGTGTA TGACCTGCTG CTGGAGATGC TGAATGCCCA CGTGCTTCGCGGGTGCAAGT 1380 CCTCCATCAC GGGGTCCGAG TGCAGCCCGG CAGAGGACAG TAAAAGCAAAGAGGGCTCCC 1440 AGAACCTACA GTCTCAGTGA 1460 485 amino acids amino acidlinear Mus musculus 5 Met Ala Phe Tyr Ser Pro Ala Val Met Asn Tyr SerVal Pro Ser Ser 1 5 10 15 Thr Gly Asn Leu Glu Gly Gly Pro Val Arg GlnThr Ala Ser Pro Asn 20 25 30 Val Leu Trp Pro Thr Ser Gly His Leu Ser ProLeu Ala Thr His Cys 35 40 45 Gln Ser Ser Leu Leu Tyr Ala Glu Pro Gln LysSer Pro Trp Cys Glu 50 55 60 Ala Arg Ser Leu Glu His Thr Leu Pro Val AsnArg Glu Thr Leu Lys 65 70 75 80 Arg Lys Leu Gly Gly Ser Gly Cys Ala SerPro Val Thr Ser Pro Ser 85 90 95 Thr Lys Arg Asp Ala His Phe Cys Ala ValCys Ser Asp Tyr Ala Ser 100 105 110 Gly Tyr His Tyr Gly Val Trp Ser CysGlu Gly Cys Lys Ala Phe Phe 115 120 125 Lys Arg Ser Ile Gln Gly His AsnAsp Tyr Ile Cys Pro Ala Thr Asn 130 135 140 Gln Cys Thr Ile Asp Lys AsnArg Arg Lys Asn Cys Gln Ala Cys Arg 145 150 155 160 Leu Arg Lys Cys TyrGlu Val Gly Met Val Lys Cys Gly Ser Arg Arg 165 170 175 Glu Arg Cys GlyTyr Arg Ile Val Arg Arg Gln Arg Ser Ala Ser Glu 180 185 190 Gln Val HisCys Leu Asn Lys Ala Lys Arg Thr Ser Gly His Thr Pro 195 200 205 Arg ValLys Glu Leu Leu Leu Asn Ser Leu Ser Pro Glu Gln Leu Val 210 215 220 LeuThr Leu Leu Glu Ala Glu Pro Pro Asn Val Leu Val Ser Arg Pro 225 230 235240 Ser Met Pro Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu 245250 255 Ala Asp Lys Glu Leu Val His Met Ile Gly Trp Ala Lys Lys Ile Pro260 265 270 Gly Phe Val Glu Leu Ser Leu Leu Asp Gln Val Arg Leu Leu GluSer 275 280 285 Cys Trp Met Glu Val Leu Met Val Gly Leu Met Trp Arg SerIle Asp 290 295 300 His Pro Gly Lys Leu Ile Phe Ala Pro Asp Leu Val LeuAsp Arg Asp 305 310 315 320 Glu Gly Lys Cys Val Glu Gly Ile Leu Glu IlePhe Asp Met Leu Leu 325 330 335 Ala Thr Thr Ala Arg Phe Arg Glu Leu LysLeu Gln His Lys Glu Tyr 340 345 350 Leu Cys Val Lys Ala Met Ile Leu LeuAsn Ser Ser Met Tyr His Leu 355 360 365 Ala Thr Ala Ser Gln Glu Ala GluSer Ser Arg Lys Leu Thr His Leu 370 375 380 Leu Asn Ala Val Thr Asp AlaLeu Val Trp Val Ile Ser Lys Ser Arg 385 390 395 400 Ile Ser Ser Gln GlnGln Ser Val Arg Leu Ala Asn Leu Leu Met Leu 405 410 415 Leu Ser His ValArg His Ile Ser Asn Lys Gly Met Glu His Leu Leu 420 425 430 Ser Met LysCys Lys Asn Val Val Pro Val Tyr Asp Leu Leu Leu Glu 435 440 445 Met LeuAsn Ala His Thr Leu Arg Gly Tyr Lys Ser Ser Ile Ser Gly 450 455 460 SerGly Cys Cys Ser Thr Glu Asp Ser Lys Ser Lys Glu Gly Ser Gln 465 470 475480 Asn Leu Gln Ser Gln 485 1458 base pairs nucleic acid double linearMus musculus 6 ATGGCATTCT ACAGTCCTGC TGTGATGAAC TACAGTGTTC CCAGCAGCACCGGTAACCTG 60 GAAGGTGGGC CTGTTCGCCA GACTGCAAGC CCAAATGTGC TATGGCCAACTTCTGGACAC 120 CTCTCTCCTT TAGCCACCCA CTGCCAATCA TCGCTTCTCT ATGCAGAACCTCAAAAGAGT 180 CCTTGGTGTG AAGCAAGATC ACTAGAACAC ACCTTGCCTG TAAACAGAGAGACCCTGAAG 240 AGGAAGCTTG GCGGGAGCGG TTGTGCCAGC CCTGTTACTA GTCCAAGCACCAAGAGGGAT 300 GCTCACTTCT GTGCCGTCTG CAGTGATTAT GCATCTGGGT ATCATTACGGTGTCTGGTCC 360 TGTGAAGGAT GTAAGGCCTT TTTTAAAAGA AGCATTCAAG GACATAATGACTATATCTGT 420 CCAGCCACGA ATCAGTGTAC GATAGACAAG AACCGGCGTA AAAACTGCCAGGCCTGCCGA 480 CTTCGCAAGT GTTACGAAGT AGGAATGGTC AAGTGTGGAT CCAGGAGAGAAAGGTGTGGG 540 TACCGAATAG TACGAAGACA GAGAAGTGCC AGCGAGCAGG TGCATTGCCTGAACAAAGCC 600 AAGAGAACCA GTGGGCACAC ACCCCGGGTG AAGGAGCTAC TGCTGAACTCTCTGAGTCCC 660 GAGCAGCTGG TGCTCACCCT GCTGGAAGCT GAGCCACCCA ATGTGCTAGTGAGTCGTCCC 720 AGCATGCCCT TCACCGAGGC CTCCATGATG ATGTCCCTTA CGAAGCTGGCTGACAAGGAA 780 CTGGTGCACA TGATTGGCTG GGCCAAGAAA ATCCCTGGCT TTGTGGAGCTCAGCCTGTTG 840 GACCAAGTCC GCCTCTTGGA AAGCTGCTGG ATGGAGGTGC TGATGGTGGGGCTGATGTGG 900 CGCTCCATCG ACCACCCCGG CAAGCTCATC TTTGCTCCAG ACCTCGTTCTGGACAGGGAT 960 GAGGGGAAGT GCGTGGAAGG GATTCTGGAA ATCTTTGACA TGCTCCTGGCGACGACGGCA 1020 CGGTTCCGTG AGTTAAAACT GCAGCACAAA GAATATCTGT GTGTGAAGGCCATGATTCTC 1080 CTCAACTCCA GTATGTACCA CTTGGCTACC GCAAGCCAGG AAGCAGAGAGTAGCCGGAAG 1140 CTGACACACC TATTGAACGC AGTGACAGAT GCCCTGGTCT GGGTGATTTCGAAGAGTAGA 1200 ATCTCTTCCC AGCAGCAGTC AGTCCGTCTG GCCAACCTCC TGATGCTTCTTTCTCATGTC 1260 AGGCACATCA GTAACAAGGG CATGGAACAT CTGCTCAGCA TGAAGTGCAAAAATGTGGTC 1320 CCGGTGTACG ACCTGCTGCT GGAGATGCTG AATGCTCACA CGCTTCGAGGGTACAAGTCC 1380 TCAATCTCGG GGTCTGGGTG CTGCTCGACA GAGGACAGTA AGAGCAAAGAGGGCTCCCAG 1440 AACCTCCAGT CTCAGTGA 1458 226 amino acids amino acidlinear Rattus rattus 7 Glu Leu Val His Met Ile Gly Trp Ala Lys Lys IlePro Gly Phe Val 1 5 10 15 Glu Leu Ser Leu Leu Asp Gln Val Arg Leu LeuGlu Ser Cys Trp Met 20 25 30 Glu Val Leu Met Val Gly Leu Met Trp Arg SerIle Asp His Pro Gly 35 40 45 Lys Leu Ile Phe Ala Pro Asp Leu Val Leu AspArg Asp Glu Gly Lys 50 55 60 Cys Val Glu Gly Ile Leu Glu Ile Phe Asp MetLeu Leu Ala Thr Thr 65 70 75 80 Ser Arg Phe Arg Glu Leu Lys Leu Gln HisLys Glu Tyr Leu Cys Val 85 90 95 Lys Ala Met Ile Leu Leu Asn Ser Ser MetTyr Pro Leu Ala Ser Ala 100 105 110 Asn Gln Glu Ala Glu Ser Ser Arg LysLeu Thr His Leu Leu Asn Ala 115 120 125 Val Thr Asp Ala Leu Val Trp ValIle Ala Lys Ser Gly Ile Ser Ser 130 135 140 Gln Gln Gln Ser Val Arg LeuAla Asn Leu Leu Met Leu Leu Ser His 145 150 155 160 Val Arg His Ile SerAsn Lys Gly Met Glu His Leu Leu Ser Met Lys 165 170 175 Cys Lys Asn ValVal Pro Val Tyr Asp Leu Leu Leu Glu Met Leu Asn 180 185 190 Ala His ThrLeu Arg Gly Tyr Lys Ser Ser Ile Ser Gly Ser Glu Cys 195 200 205 Ser SerThr Glu Asp Ser Lys Asn Lys Glu Ser Ser Gln Asn Leu Gln 210 215 220 SerGln 225 243 amino acids amino acid linear Rattus rattus 8 Glu Leu ValHis Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Gly 1 5 10 15 Asp LeuAsn Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 20 25 30 Glu IleLeu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly 35 40 45 Lys LeuLeu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 50 55 60 Cys ValGlu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 65 70 75 80 SerArg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 85 90 95 LysSer Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 100 105 110Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 115 120125 Lys Ile Asn Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 130135 140 Leu Gln Gln Gln His Arg Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser145 150 155 160 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu TyrAsn Met 165 170 175 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu LeuGlu Met Leu 180 185 190 Asp Ala His Arg Leu His Ala Pro Ala Ser Arg MetGly Val Pro Pro 195 200 205 Glu Glu Pro Ser Gln Ser Gln Leu Thr Thr ThrSer Ser Thr Ser Ala 210 215 220 His Ser Leu Gln Thr Tyr Tyr Ile Pro ProGlu Ala Glu Gly Phe Pro 225 230 235 240 Asn Thr Ile 243 amino acidsamino acid linear Mus musculus 9 Glu Leu Val His Met Ile Asn Trp Ala LysArg Val Pro Gly Phe Gly 1 5 10 15 Asp Leu Asn Leu His Asp Gln Val HisLeu Leu Glu Cys Ala Trp Leu 20 25 30 Glu Ile Leu Met Ile Gly Leu Val TrpArg Ser Met Glu His Pro Gly 35 40 45 Lys Leu Leu Phe Ala Pro Asn Leu LeuLeu Asp Arg Asn Gln Gly Lys 50 55 60 Cys Val Glu Gly Met Val Glu Ile PheAsp Met Leu Leu Ala Thr Ser 65 70 75 80 Ser Arg Phe Arg Met Met Asn LeuGln Gly Glu Glu Phe Val Cys Leu 85 90 95 Lys Ser Ile Ile Leu Leu Asn SerGly Val Tyr Thr Phe Leu Ser Ser 100 105 110 Thr Leu Lys Ser Leu Glu GluLys Asp His Ile His Arg Val Leu Asp 115 120 125 Lys Ile Thr Asp Thr LeuIle His Leu Met Ala Lys Ala Gly Leu Thr 130 135 140 Leu Gln Gln Gln HisArg Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 145 150 155 160 His Ile ArgHis Met Ser Asn Lys Gly Met Glu His Leu Tyr Asn Met 165 170 175 Lys CysLys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 180 185 190 AspAla His Arg Leu His Ala Pro Ala Ser Arg Met Gly Val Pro Pro 195 200 205Glu Glu Pro Ser Gln Thr Gln Leu Ala Thr Thr Ser Ser Thr Ser Ala 210 215220 His Ser Leu Gln Thr Tyr Tyr Ile Pro Pro Glu Ala Glu Gly Phe Pro 225230 235 240 Asn Thr Ile 243 amino acids amino acid linear Homo sapiens10 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 1 510 15 Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 2025 30 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Val 3540 45 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 5055 60 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 6570 75 80 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu85 90 95 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser100 105 110 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val LeuAsp 115 120 125 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala GlyLeu Thr 130 135 140 Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu LeuIle Leu Ser 145 150 155 160 His Ile Arg His Met Ser Asn Lys Gly Met GluHis Leu Tyr Ser Met 165 170 175 Lys Cys Lys Asn Val Val Pro Leu Tyr AspLeu Leu Leu Glu Met Leu 180 185 190 Asp Ala His Arg Leu His Ala Pro ThrSer Arg Gly Gly Ala Ser Val 195 200 205 Glu Glu Thr Asp Gln Ser His LeuAla Thr Ala Gly Ser Thr Ser Ser 210 215 220 His Ser Leu Gln Lys Tyr TyrIle Thr Gly Glu Ala Glu Gly Phe Pro 225 230 235 240 Ala Thr Val 66 aminoacids amino acid linear Rattus rattus 11 Cys Pro Val Cys Ser Asp Tyr AlaSer Gly Tyr His Tyr Gly Val Trp 1 5 10 15 Ser Cys Glu Gly Cys Lys AlaPhe Phe Lys Arg Ser Ile Gln Gly His 20 25 30 Asn Asp Tyr Ile Cys Pro AlaThr Asn Gln Cys Thr Ile Asp Lys Asn 35 40 45 Arg Arg Lys Ser Cys Gln AlaCys Arg Leu Arg Lys Cys Tyr Glu Val 50 55 60 Gly Met 65 66 amino acidsamino acid linear 12 Cys Ala Val Cys Asn Asp Tyr Ala Ser Gly Tyr His TyrGly Val Trp 1 5 10 15 Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg SerIle Gln Gly His 20 25 30 Asn Asp Tyr Met Cys Pro Ala Thr Asn Gln Cys ThrIle Asp Lys Asn 35 40 45 Arg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg LysCys Tyr Glu Val 50 55 60 Gly Met 65 484 amino acids amino acid linearRattus rattus 13 Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Val ProGly Ser 1 5 10 15 Thr Ser Asn Leu Asp Gly Gly Pro Val Arg Leu Ser ThrSer Pro Asn 20 25 30 Val Leu Trp Pro Thr Ser Gly His Leu Ser Pro Leu AlaThr His Cys 35 40 45 Gln Ser Ser Leu Leu Tyr Ala Glu Pro Gln Lys Ser ProTrp Cys Glu 50 55 60 Ala Arg Ser Leu Glu His Thr Leu Pro Val Asn Arg GluThr Leu Lys 65 70 75 80 Arg Lys Leu Ser Gly Ser Ser Cys Ala Ser Pro ValThr Ser Pro Asn 85 90 95 Ala Lys Arg Asp Ala His Phe Cys Pro Val Cys SerAsp Tyr Ala Ser 100 105 110 Gly Tyr His Tyr Gly Val Trp Ser Cys Glu GlyCys Lys Ala Phe Phe 115 120 125 Lys Arg Ser Ile Gln Gly His Asn Asp TyrIle Cys Pro Ala Thr Asn 130 135 140 Gln Cys Thr Ile Asp Lys Asn Arg ArgLys Ser Cys Gln Ala Cys Arg 145 150 155 160 Leu Arg Lys Cys Tyr Glu ValGly Met Val Lys Cys Gly Ser Arg Arg 165 170 175 Glu Arg Cys Gly Tyr ArgIle Val Arg Arg Gln Arg Ser Ser Ser Glu 180 185 190 Gln Val His Cys LeuSer Lys Ala Lys Arg Asn Gly Gly His Ala Pro 195 200 205 Arg Val Lys GluLeu Leu Leu Ser Thr Leu Ser Pro Glu Gln Leu Val 210 215 220 Leu Thr LeuLeu Glu Ala Glu Pro Pro Asn Val Leu Val Ser Arg Pro 225 230 235 240 SerMet Pro Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu 245 250 255Ala Asp Lys Glu Leu Val His Met Ile Gly Trp Ala Lys Lys Ile Pro 260 265270 Gly Phe Val Glu Leu Ser Leu Leu Asp Gln Val Arg Leu Leu Glu Ser 275280 285 Cys Trp Met Glu Val Leu Met Val Gly Leu Met Trp Arg Ser Ile Asp290 295 300 His Pro Gly Lys Leu Ile Phe Ala Pro Asp Leu Val Leu Asp ArgAsp 305 310 315 320 Glu Gly Lys Cys Val Glu Gly Ile Leu Glu Ile Phe AspMet Leu Leu 325 330 335 Ala Thr Thr Ser Arg Phe Arg Glu Leu Lys Leu GlnHis Lys Glu Tyr 340 345 350 Leu Cys Val Lys Ala Met Ile Leu Leu Asn SerSer Met Tyr Pro Leu 355 360 365 Ala Ser Ala Asn Gln Glu Ala Glu Ser SerArg Lys Leu Thr His Leu 370 375 380 Leu Asn Ala Val Thr Asp Ala Leu ValTrp Val Ile Ala Lys Ser Gly 385 390 395 400 Ile Ser Ser Gln Gln Gln SerVal Arg Leu Ala Asn Leu Leu Met Leu 405 410 415 Leu Ser His Val Arg HisIle Ser Asn Lys Gly Met Glu His Leu Leu 420 425 430 Ser Met Lys Cys LysAsn Val Val Pro Val Tyr Asp Leu Leu Leu Glu 435 440 445 Met Leu Asn AlaHis Thr Leu Arg Gly Tyr Lys Ser Ser Ile Ser Gly 450 455 460 Ser Glu CysSer Ser Thr Glu Asp Ser Lys Asn Lys Glu Ser Ser Gln 465 470 475 480 AsnLeu Gln Ser 484 amino acids amino acid linear Mus musculus 14 Met AlaPhe Tyr Ser Pro Ala Val Met Asn Tyr Ser Val Pro Ser Ser 1 5 10 15 ThrGly Asn Leu Glu Gly Gly Pro Val Arg Gln Thr Ala Ser Pro Asn 20 25 30 ValLeu Trp Pro Thr Ser Gly His Leu Ser Pro Leu Ala Thr His Cys 35 40 45 GlnSer Ser Leu Leu Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu 50 55 60 AlaArg Ser Leu Glu His Thr Leu Pro Val Asn Arg Glu Thr Leu Lys 65 70 75 80Arg Lys Leu Gly Gly Ser Gly Cys Ala Ser Pro Val Thr Ser Pro Ser 85 90 95Thr Lys Arg Asp Ala His Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser 100 105110 Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe 115120 125 Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn130 135 140 Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Asn Cys Gln Ala CysArg 145 150 155 160 Leu Arg Lys Cys Tyr Glu Val Gly Met Val Lys Cys GlySer Arg Arg 165 170 175 Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Gln ArgSer Ala Ser Glu 180 185 190 Gln Val His Cys Leu Asn Lys Ala Lys Arg ThrSer Gly His Thr Pro 195 200 205 Arg Val Lys Glu Leu Leu Leu Asn Ser LeuSer Pro Glu Gln Leu Val 210 215 220 Leu Thr Leu Leu Glu Ala Glu Pro ProAsn Val Leu Val Ser Arg Pro 225 230 235 240 Ser Met Pro Phe Thr Glu AlaSer Met Met Met Ser Leu Thr Lys Leu 245 250 255 Ala Asp Lys Glu Leu ValHis Met Ile Gly Trp Ala Lys Lys Ile Pro 260 265 270 Gly Phe Val Glu LeuSer Leu Leu Asp Gln Val Arg Leu Leu Glu Ser 275 280 285 Cys Trp Met GluVal Leu Met Val Gly Leu Met Trp Arg Ser Ile Asp 290 295 300 His Pro GlyLys Leu Ile Phe Ala Pro Asp Leu Val Leu Asp Arg Asp 305 310 315 320 GluGly Lys Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Leu 325 330 335Ala Thr Thr Ala Arg Phe Arg Glu Leu Lys Leu Gln His Lys Glu Tyr 340 345350 Leu Cys Val Lys Ala Met Ile Leu Leu Asn Ser Ser Met Tyr His Leu 355360 365 Ala Thr Ala Ser Gln Glu Ala Glu Ser Ser Arg Lys Leu Thr His Leu370 375 380 Leu Asn Ala Val Thr Asp Ala Leu Val Trp Val Ile Ser Lys SerArg 385 390 395 400 Ile Ser Ser Gln Gln Gln Ser Val Arg Leu Ala Asn LeuLeu Met Leu 405 410 415 Leu Ser His Val Arg His Ile Ser Asn Lys Gly MetGlu His Leu Leu 420 425 430 Ser Met Lys Cys Lys Asn Val Val Pro Val TyrAsp Leu Leu Leu Glu 435 440 445 Met Leu Asn Ala His Thr Leu Arg Gly TyrLys Ser Ser Ile Ser Gly 450 455 460 Ser Gly Cys Cys Ser Thr Glu Asp SerLys Ser Lys Glu Gly Ser Gln 465 470 475 480 Asn Leu Gln Ser 384 aminoacids amino acid linear Homo sapiens 15 Ala Leu Ser Pro Leu Val Val HisArg Gln Leu Ser His Leu Tyr Ala 1 5 10 15 Glu Pro Gln Lys Ser Pro TrpCys Glu Ala Arg Ser Leu Glu His Thr 20 25 30 Leu Pro Val Asn Arg Glu ThrLeu Lys Arg Lys Val Ser Gly Asn Arg 35 40 45 Cys Ala Ser Pro Val Thr GlyPro Gly Ser Lys Arg Asp Ala His Phe 50 55 60 Cys Ala Val Cys Ser Asp TyrAla Ser Gly Tyr His Tyr Gly Val Trp 65 70 75 80 Ser Cys Glu Gly Cys LysAla Phe Phe Lys Arg Ser Ile Gln Gly His 85 90 95 Asn Asp Tyr Ile Cys ProAla Thr Asn Gln Cys Thr Ile Asp Lys Asn 100 105 110 Arg Arg Lys Ser CysGln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val 115 120 125 Gly Met Val LysCys Gly Ser Arg Arg Glu Arg Cys Gly Tyr Arg Leu 130 135 140 Val Arg ArgGln Arg Ser Ala Asp Glu Gln Leu His Cys Ala Gly Lys 145 150 155 160 AlaLys Arg Ser Gly Gly His Ala Pro Arg Val Arg Glu Leu Leu Leu 165 170 175Asp Ala Leu Ser Pro Glu Gln Leu Val Leu Thr Leu Leu Glu Ala Glu 180 185190 Pro Pro His Val Leu Ile Ser Arg Pro Ser Ala Pro Phe Thr Glu Ala 195200 205 Ser Met Met Met Ser Leu Thr Lys Leu Ala Asp Lys Glu Leu Val His210 215 220 Met Ile Ser Trp Ala Lys Lys Ile Pro Gly Phe Val Glu Leu SerLeu 225 230 235 240 Phe Asp Gln Val Arg Leu Leu Glu Ser Cys Trp Met GluVal Leu Met 245 250 255 Met Gly Leu Met Trp Arg Ser Ile Asp His Pro GlyLys Leu Ile Phe 260 265 270 Ala Pro Asp Leu Val Leu Asp Arg Asp Glu GlyLys Cys Val Glu Gly 275 280 285 Ile Leu Glu Ile Phe Asp Met Leu Leu AlaThr Thr Ser Arg Phe Arg 290 295 300 Glu Leu Lys Leu Gln His Lys Glu TyrLeu Cys Val Lys Ala Met Ile 305 310 315 320 Leu Leu Asn Ser Ser Met TyrPro Leu Val Thr Ala Thr Gln Asp Ala 325 330 335 Asp Ser Ser Arg Lys LeuAla His Leu Leu Asn Ala Val Thr Asp Ala 340 345 350 Leu Val Trp Val IleAla Lys Ser Gly Ile Ser Ser Gln Gln Gln Ser 355 360 365 Met Arg Leu AlaAsn Leu Leu Met Leu Leu Ser His Val Arg His Ala 370 375 380 596 aminoacids amino acid linear Rattus rattus 16 Met Thr Met Thr Leu His Thr LysAla Ser Gly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu LeuGlu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 Met Pro Met Glu Arg Ala LeuGly Glu Val Tyr Val Asp Asn Ser Lys 35 40 45 Pro Ala Val Phe Asn Tyr ProGlu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Ala Ala AlaGly Ala Ser Ala Pro Val Tyr Gly Gln 65 70 75 80 Ser Ser Ile Thr Tyr GlyPro Gly Ser Glu Ala Ala Ala Phe Gly Ala 85 90 95 Asn Ser Leu Gly Ala PhePro Gln Leu Asn Ser Val Ser Pro Ser Pro 100 105 110 Ile Met Ile Leu HisPro Pro Pro His Val Ser Pro Phe Leu His Pro 115 120 125 His Gly His GlnVal Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Ala Tyr 130 135 140 Ala Val ArgAsp Thr Gly Pro Pro Ala Phe Tyr Arg Ser Asn Ser Asp 145 150 155 160 AsnArg Arg Gln Asn Gly Arg Glu Arg Leu Ser Ser Ser Ser Glu Lys 165 170 175Gly Asn Met Ile Met Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val 180 185190 Cys Asn Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu 195200 205 Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr210 215 220 Met Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp Lys Asn Arg ArgLys 225 230 235 240 Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu ValGly Met Met 245 250 255 Lys Gly Gly Ile Arg Lys Asp Arg Arg Gly Gly ArgMet Leu Lys His 260 265 270 Lys Arg Gln Arg Asp Asp Leu Glu Gly Arg AsnGlu Met Gly Thr Ser 275 280 285 Gly Asp Met Arg Ala Ala Asn Leu Trp ProSer Pro Leu Val Ile Lys 290 295 300 His Thr Lys Lys Asn Ser Pro Ala LeuSer Leu Thr Ala Asp Gln Met 305 310 315 320 Val Ser Ala Leu Leu Asp AlaGlu Pro Pro Leu Ile Tyr Ser Glu Tyr 325 330 335 Asp Pro Ser Arg Pro PheSer Glu Ala Ser Met Met Gly Leu Leu Thr 340 345 350 Asn Leu Ala Asp ArgGlu Leu Val His Met Ile Asn Trp Ala Lys Arg 355 360 365 Val Pro Gly PheGly Asp Leu Asn Leu His Asp Gln Val His Leu Leu 370 375 380 Glu Cys AlaTrp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser 385 390 395 400 MetGlu His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp 405 410 415Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met 420 425430 Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu 435440 445 Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr450 455 460 Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp HisIle 465 470 475 480 His Arg Val Leu Asp Lys Ile Asn Asp Thr Leu Ile HisLeu Met Ala 485 490 495 Lys Ala Gly Leu Thr Leu Gln Gln Gln His Arg ArgLeu Ala Gln Leu 500 505 510 Leu Leu Ile Leu Ser His Ile Arg His Met SerAsn Lys Gly Met Glu 515 520 525 His Leu Tyr Asn Met Lys Cys Lys Asn ValVal Pro Leu Tyr Asp Leu 530 535 540 Leu Leu Glu Met Leu Asp Ala His ArgLeu His Ala Pro Ala Ser Arg 545 550 555 560 Met Gly Val Pro Pro Glu GluPro Ser Gln Ser Gln Leu Thr Thr Thr 565 570 575 Ser Ser Thr Ser Ala HisSer Leu Gln Thr Tyr Tyr Ile Pro Pro Glu 580 585 590 Ala Glu Gly Phe 595591 amino acids amino acid linear Homo sapiens 17 Met Thr Met Thr LeuHis Thr Lys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln GlyAsn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu GluArg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val TyrAsn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala AlaAsn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro GlySer Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro ProLeu Asn Ser Val Ser Pro Ser Pro Ile Met Ile Leu His 100 105 110 Pro ProPro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125 ProTyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150155 160 Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met165 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp TyrAla 180 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys LysAla Phe 195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met CysPro Ala Thr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys SerCys Gln Ala Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly MetMet Lys Gly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met LeuLys His Lys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly Arg Gly Glu ValGly Ser Ala Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu Trp Pro Ser ProLeu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu Ala Leu Ser LeuThr Ala Asp Gln Met Val Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu ProPro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser GluAla Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu LeuVal His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365 AspLeu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Val 385 390395 400 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys405 410 415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala ThrSer 420 425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe ValCys Leu 435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr PheLeu Ser Ser 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile HisArg Val Leu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu MetAla Lys Ala Gly Leu Thr 485 490 495 Leu Gln Gln Gln His Gln Arg Leu AlaGln Leu Leu Leu Ile Leu Ser 500 505 510 His Ile Arg His Met Ser Asn LysGly Met Glu His Leu Tyr Ser Met 515 520 525 Lys Cys Lys Asn Val Val ProLeu Tyr Asp Leu Leu Leu Glu Met Leu 530 535 540 Asp Ala His Arg Leu HisAla Pro Thr Ser Arg Gly Gly Ala Ser Val 545 550 555 560 Glu Glu Thr AspGln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575 His Ser LeuGln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe 580 585 590 518 aminoacids amino acid linear Homo sapiens 18 Met Gly Leu Glu Met Ser Ser LysAsp Ser Pro Gly Ser Leu Asp Gly 1 5 10 15 Arg Ala Trp Glu Asp Ala GlnLys Pro Gln Ser Ala Trp Cys Gly Gly 20 25 30 Arg Lys Thr Arg Val Tyr AlaThr Ser Ser Arg Arg Ala Pro Pro Ser 35 40 45 Glu Gly Thr Arg Arg Gly GlyAla Ala Arg Pro Glu Glu Ala Ala Glu 50 55 60 Glu Gly Pro Pro Ala Ala ProGly Ser Leu Arg His Ser Gly Pro Leu 65 70 75 80 Gly Pro His Ala Cys ProThr Ala Leu Pro Glu Pro Gln Val Thr Ser 85 90 95 Ala Met Ser Ser Gln ValVal Gly Ile Glu Pro Leu Tyr Ile Lys Ala 100 105 110 Glu Pro Ala Ser ProAsp Ser Pro Lys Gly Ser Ser Glu Thr Glu Thr 115 120 125 Glu Pro Pro ValAla Leu Ala Pro Gly Pro Ala Pro Thr Arg Cys Leu 130 135 140 Pro Gly HisLys Glu Glu Glu Asp Gly Glu Gly Ala Gly Pro Gly Glu 145 150 155 160 GlnGly Gly Gly Lys Leu Val Leu Ser Ser Leu Pro Lys Arg Leu Cys 165 170 175Leu Val Cys Gly Asp Val Ala Ser Gly Tyr His Tyr Gly Val Ala Ser 180 185190 Cys Glu Ala Cys Lys Ala Phe Phe Lys Arg Thr Ile Gln Gly Ser Ile 195200 205 Glu Tyr Ser Cys Pro Ala Ser Asn Glu Cys Glu Ile Thr Lys Arg Arg210 215 220 Arg Lys Ala Cys Gln Ala Cys Arg Phe Thr Lys Cys Ile Arg ValGly 225 230 235 240 Met Leu Lys Glu Gly Val Arg Leu Asp Arg Val Arg GlyGly Arg Gln 245 250 255 Lys Tyr Lys Arg Arg Pro Glu Val Asp Pro Leu ProPhe Pro Gly Pro 260 265 270 Phe Pro Ala Gly Pro Leu Ala Val Ala Gly GlyPro Arg Lys Thr Ala 275 280 285 Ala Pro Val Asn Ala Leu Val Ser His LeuLeu Val Val Glu Pro Glu 290 295 300 Lys Leu Tyr Ala Met Pro Asp Pro AlaGly Pro Asp Gly His Leu Pro 305 310 315 320 Ala Val Ala Thr Leu Cys AspLeu Phe Asp Arg Glu Ile Val Val Thr 325 330 335 Ile Ser Trp Ala Lys SerIle Pro Gly Phe Ser Ser Leu Ser Leu Ser 340 345 350 Asp Gln Met Ser ValLeu Gln Ser Val Trp Met Glu Val Leu Val Leu 355 360 365 Gly Val Ala GlnArg Ser Leu Pro Leu Gln Asp Glu Leu Ala Phe Ala 370 375 380 Glu Asp LeuVal Leu Ile Glu Glu Gly Ala Arg Ala Ala Gly Leu Gly 385 390 395 400 GluLeu Gly Ala Ala Leu Leu Gln Leu Val Arg Arg Leu Gln Ala Leu 405 410 415Arg Leu Glu Arg Glu Glu Tyr Val Leu Leu Lys Ala Leu Ala Leu Ala 420 425430 Asn Ser Asp Ser Val His Ile Glu Asp Glu Pro Arg Leu Trp Ser Ser 435440 445 Cys Glu Lys Leu Leu His Glu Ala Leu Leu Glu Tyr Glu Ala Gly Arg450 455 460 Ala Gly Pro Gly Gly Gly Ala Glu Arg Arg Arg Ala Gly Arg LeuLeu 465 470 475 480 Leu Thr Leu Pro Leu Leu Arg Gln Thr Ala Gly Lys ValLeu Ala His 485 490 495 Phe Tyr Gly Val Lys Leu Glu Gly Lys Val Pro MetHis Lys Leu Phe 500 505 510 Leu Glu Met Leu Glu Ala 515 431 amino acidsamino acid linear Homo sapiens 19 Met Ser Ser Glu Asp Arg His Leu GlySer Ser Cys Gly Ser Phe Ile 1 5 10 15 Lys Thr Glu Pro Ser Ser Pro SerSer Gly Ile Asp Ala Leu Ser His 20 25 30 His Ser Pro Ser Gly Ser Ser AspAla Ser Gly Gly Phe Gly Met Ala 35 40 45 Leu Gly Thr His Ala Asn Gly LeuAsp Ser Pro Pro Met Phe Ala Gly 50 55 60 Ala Gly Leu Gly Gly Asn Pro CysArg Lys Ser Tyr Glu Asp Cys Thr 65 70 75 80 Ser Gly Ile Met Glu Asp SerAla Ile Lys Cys Glu Tyr Met Leu Asn 85 90 95 Ala Ile Pro Lys Arg Leu CysLeu Val Cys Gly Asp Ile Ala Ser Gly 100 105 110 Tyr His Tyr Gly Val AlaSer Cys Glu Ala Cys Lys Ala Phe Phe Lys 115 120 125 Arg Thr Ile Gln GlyAsn Ile Glu Tyr Ser Cys Pro Ala Thr Asn Glu 130 135 140 Cys Glu Ile ThrLys Arg Arg Arg Lys Ser Cys Gln Ala Cys Arg Phe 145 150 155 160 Met LysCys Ile Lys Val Gly Met Leu Lys Glu Gly Val Arg Leu Asp 165 170 175 ArgVal Arg Gly Gly Arg Gln Lys Tyr Lys Arg Arg Leu Asp Ser Glu 180 185 190Asn Ser Pro Tyr Leu Ser Leu Gln Ile Ser Pro Pro Ala Lys Lys Pro 195 200205 Leu Thr Lys Ile Val Ser Tyr Leu Leu Val Ala Glu Pro Asp Lys Leu 210215 220 Tyr Ala Met Pro Pro Asp Asp Val Pro Glu Gly Asp Ile Lys Ala Leu225 230 235 240 Thr Thr Leu Cys Asp Leu Ala Asp Arg Glu Leu Val Phe LeuIle Ser 245 250 255 Trp Ala Lys His Ile Pro Gly Phe Ser Asn Leu Thr LeuGly Asp Gln 260 265 270 Met Ser Leu Leu Gln Ser Ala Trp Met Glu Ile LeuIle Leu Gly Ile 275 280 285 Val Tyr Arg Ser Leu Pro Tyr Asp Asp Lys LeuAla Tyr Ala Glu Asp 290 295 300 Tyr Ile Met Asp Glu Glu His Ser Arg LeuVal Gly Leu Leu Glu Leu 305 310 315 320 Tyr Arg Ala Ile Leu Gln Leu ValArg Arg Tyr Lys Lys Leu Lys Val 325 330 335 Glu Lys Glu Glu Phe Val MetLeu Lys Ala Ile Ala Leu Ala Asn Ser 340 345 350 Asp Ser Met Tyr Ile GluAsn Leu Glu Ala Val Gln Lys Leu Gln Asp 355 360 365 Leu Leu His Glu AlaLeu Gln Asp Tyr Glu Leu Ser Gln Arg His Glu 370 375 380 Glu Pro Arg ArgAla Gly Lys Leu Leu Leu Thr Leu Pro Leu Leu Arg 385 390 395 400 Gln ThrAla Ala Lys Ala Val Gln His Phe Tyr Ser Val Lys Leu Gln 405 410 415 GlyLys Val Pro Met His Lys Leu Phe Leu Glu Met Leu Glu Ala 420 425 430

1. A receptor, ERβ, having the amino acid sequence of FIGS. 1, 13A or14A or substantially the same amino acid sequence as the amino acidsequence shown in FIGS. 1, 13A or 14A or an amino acid sequencefunctionally similar to those sequences.
 2. A receptor according toclaim 1 having an amino acid sequence which is more than 95%, identicalwith the sequence shown in FIGS. 1, 13A or 14A.
 3. A receptor accordingto claim 1 or 2 which is derived from rat or human cells.
 4. A receptoraccording to claim 1, 2 or 3 which is an estrogen receptor.
 5. A DNAsequence encoding a receptor according to claim 1, 2, 3, or
 4. 6. A DNAsequence according to claim 5 in which the DNA sequence is that given inFIGS. 1, 13B or 14B or is a DNA sequence encoding a protein orpolypeptide having the functionally of ERβ.
 7. The use of a receptoraccording to claim 1, 2, 3 or 4 or a DNA sequence according to claim 5or 6 in an assay to determine molecules which bind Erβ.
 8. The use of areceptor according to claim 1, 2, 3 or 4 or a DNA sequence according toclaim 5 or 6 in an assay to determine molecules for use in the treatmentof Erα or Erβ specific diseases or conditions.
 9. The use of a receptoraccording to claim 1, 2, 3 or 4 or a DNA sequence according to claim 5or 6 in an assay to determine molecules for use in treatment of prostateor ovarian cancer, benign prostatic hyperplasia, diseases of the centralnervous system, osteoporosis, or cardiovascular disease.
 10. A drugdesign method comprising comparing binding of a test compound to ERα andto ERβ.
 11. hERβ and functional equivalents thereof.
 12. The use of areceptor according to claim 1, 2, 3 or 4 in the testing of the possibleestrogenic or other hormonal effect of a substance.